Mir 101 mimic

The Jurkat and HEK293 cell lines were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA), and cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37°C in a humidified atmosphere with 5% CO₂. Adriamycin (ADM) was obtained from Sangon Biotech Co., Ltd., (Shanghai, China) and dissolved in phosphate-buffered saline (PBS). Cells were treated with 5 µg/ml adriamycin for 24 h. The cells were transfected with miR-negative control (NC), miR-101 mimic, miR-101 inhibitor, Notch1-pcDNA3.1 or pcDNA3.1 empty vector (Shanghai GenePharma, Co., Ltd., Shanghai, China) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) following the manufacturer's protocols. After 6 h, the medium was replaced with fresh medium for further experiments.

The miR-101-mimic, control-mimic, inhibitor and inhibitor-control were designed and synthesized by Shanghai GenePharma Co., Ltd. miRNA (~50 nM) was transfected using Lipofectamine™ 2000 (Invitrogen) as recommended by the manufacturer. After 48 h of transfection, total RNA was extracted and the efficiency of transfection was verified by reverse transcription-quantitative PCR (RT-qPCR). For in vivo chemosensitivity assays, miR-101 mimic, control-mimic, miR-101 inhibitor and inhibitor-control sequences were embedded into the lentiviral pGIPZ plasmid (Genechem, Inc.). HepG2 cells were subsequently infected with the lentiviruses and inoculated into mice to produce tumor xenografts after 24 h of transfection. miRNA sequences were as follows: miR-101 mimic sense, 5′-UACAGUACUGUGAUAACUGAA-3′ and antisense, 5′-CAGUUAUCACAGUACUGUAUU-3′; control-mimic sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; miR-101 inhibitor, 5′-UUCAGUUAUCACAGUAUGUA-3′; and inhibitor-control, 5′-CAGUACUUUUGUGUAGUACAA-3′.